In 1997, Professor Folkman from of Harvard University discovered the endogenous angiogenesis inhibitor—Endostatin (ES). Endostatin is a 20-kDa cleavage fragment of the C-terminus of collagen XVIII, which had inhibitory activities on the proliferation, migration of vascular endothelial cells, and the formation of blood vessels in vivo. The recombinant endostatin can inhibit the growth and metastasis of various types of tumors in mice, and can even cure the tumor without inducing drug resistance (Folkman J et al. Cell 1997; 88:277-285; Folkman J et al. Nature 1997; 390:404-407).
The mechanism underlying the inhibitory capacity of ES is that it suppresses the angiogenesis in tumor tissues and blocks the supply of nutrition and oxygen. In China, the recombinant human endostatin (Endu) expressed by E. coli has become an anti-tumor therapeutic and its anti-tumor effect has been widely tested in clinical trail mainly focused on non-small-cell lung carcinoma. Endu, a variant of ES, has additional amino acid sequence (MGGSHHHHH; SEQ ID NO:42) on N-terminal of ES, exhibiting more thermal dynamic stability and biological activity compared with wild type human ES expressed by yeast (Fu Y. et al. Biochemistry 2010; 49:6420-6429). Other report showed that the 27 amino acids on N-terminal of ES have the similar inhibitory activities on angiogenesis compared with the complete ES (Robert TjinThamSjin, et al., Cancer Res. 2005; 65(9):3656-63). Therefore, there are many researchers design medicaments based on the N-terminal 27 amino acids activities.
Furthermore, to prolong the half-life of ES in vivo, many molecular modifications and drug design have been made to ES, including single site or multiple sites PEG modifications and conjugation with antibody Fc fragment (Tong-Young Lee, et al., Clin Cancer Res 2008; 14(5):1487-1493). Multiple sites PEG modifications of ES are usually implemented on the amino of lysine side chain. Although this may prolong the half-life of ES, but its biological activities are apparently reduced (Guoying Mou, dissertation of Shandong University, CNKI, 2005). Compared with this modification technique, single site PEG modification on the N-terminal can not only enhance the stability, but also the biological activities of ES (CN100475270C). The related product has entered into clinical trail.
Since the discovery of ES, research projects from different laboratories focused on its tumor inhibitory activities have obtained different results. Professor Folkman's lab cured tumor in mice completely using ES (Folkman J et al., 1997, Nature, 390:404-407), but many other labs could not repeat this result (News Focus, 2002, Science, 295:2198-2199). Meanwhile, since the ES produced in the prokaryotic expressing system containing polar body that is very hard to refold, many researchers diverted to use yeast to produce resolvable ES, but this did not achieve ideal results. Subsequent studies observed that the yeast expressed ES was N-terminal truncated and the truncated forms were identified as N-1, N-3, and N-4. The integrity of N-terminal is very important to the stability and biological activity of ES, this explains the confusing results obtained from yeast expressed ES (Fu Y. et al. Biochemistry 2010; 49:6420-6429).
The primary biological function of ES is that is inhibits activities of endothelial cells, including inhibiting proliferation, migration and tube formation of endothelial cells and inducing apoptosis of endothelial cell, etc. The mechanism study of molecular function shows that nucleolin locating on the surface of plasma membrane is the functional receptor of ES and mediates the endocytosis of ES and its downstream signal pathway (Shi H B, et al., Blood, 2007, 110:2899-2906). Other report shows that nucleolin is also expressed on the plasma membrane of highly proliferative breast cancer cell line MDA-MB-435 and can mediate the endocytosis of its ligand protein in MDA-MB-435 (Sven Chridtian, et al., JBC, 2003, 163(4):871-878). In other studies, integrins, tropomyosin, glypican, laminin and matrix metalloproteinase 2 (MMP2) are all observed to be the potential receptors of ES (Sudhakar, A., et al., 2003, Proc. Natl. Acad. Sci. USA 100:4766-4771; Javaherian, K., et al., 2002, J. Biol. Chem., 277:45211-45218; Karumanchi, S., et al., 2001, Mol. Cell, 7:811-822; Lee, S. J., et al., 2002, FEBS Lett., 519:147-152; MacDonald, N. J., et al., 2001, J. Biol. Chem., 276:25190-25196; Kim, Y. M., et al., 2002, J. Biol. Chem., 277:27872-27879). Moreover, the treatment of nystatin dramatically increased the endocytosis and absorption of ES in endothelial cells, and therefore enhanced the biological activities of ES on inhibiting endothelial cells migration and animal tumor growth (Chen Y, et al., 2011, Blood, 117:6392-6403).
The classical method to detect the biological activities of ES is based on its activity of inhibiting the endothelial cells, including the inhibition of migration, proliferation and tube formation of endothelial cells and other experiments. Commonly used endothelial cells mainly comprise human vascular endothelial cells (HMEC) and human umbilical vein endothelial cells (HUVEC). However, these methods require high quality of cell culture and complicated techniques, are very subjective, and exhibit low accuracy and reproducibility (Li Y H, et al., 2011, Chin J Biological March, Vol. 24 No. 3:320-323). Therefore, to explore and develop new methods of evaluating the biological activities of ES and its mutants is of great importance in the ES drug discovery and quality control.
Adenosine triphosphate (ATP) is an essential energy supply to organisms, participating in multi physiological and biological reactions and plays an important role in maintaining normal organic activities. ATP can be produced in many cellular metabolic pathways: in the most classical pathway it is produced by adenosine triphosphate synthetase through oxidative phosphorylation in mitochondrial under normal conditions, or produced in chloroplast through photosynthesis in plant. The source for ATP synthesis is mainly glucose and fatty acid. Under normal physiological conditions, the molar concentration of ATP in cell and blood are 1-10 mM and 100 μM, respectively.
ATPase, also named adenosine triphosphotase, is an enzyme that catalyzes ATP to produce ADP and Pi and releasing energy. Under most conditions, the energy produced in this reaction can be transferred to another energy-required reaction and this process has been widely utilized in all known forms of lives. In addition, high-energy bond contained in the GTP can provide energy for protein synthesis, as well. Hsp90, myosin and other proteins all depend on ATP to perform biological activities, and thus all these proteins have ATPase activities. Although various kinds of ATPase are different in terms of sequence and tertiary structure, usually all these proteins have P-loop structure as the ATP binding motif (Andrea T. Deyrup, et al., 1998, JBC, 273(16):9450-9456). This P-loop structure exhibits the following typical sequences: GXXGXXK (SEQ ID NO:34) (Driscoll, W. J., et al., 1995 Proc. Natl. Acad. Sci. U.S.A., 92:12328-12332), (G/A)XXXXGK(T/S) (SEQ ID NO:35) (Walker, J., et al., 1982, EMBO J., 1:945-951), GXXXXGKS (SEQ ID NO:36) (Satishchandran, C., et al., 1992, Biochemistry, 31:11684-11688) and GXXGXGKS (SEQ ID NO:37) (Thomas, P. M., et al., 1995, Am. J. Hum. Genet., 59:510-518). Except for X, the remaining amino acid residues are relatively conserved. Generally, GTP also can bind to the ATP binding motif of these ATPases, and thus ATP and GTP can be alternative in many cases.
Cancer cells and highly proliferative cells including endothelial cells have abnormally strong metabolism and the metabolic pathways are greatly different from normal mature cells. On one hand, cancer cells and proliferative cells demand large amount of ATP; on the other hand, the efficacy of using glucose to produce ATP is very low in these cells. This is because most cancer cells and highly proliferative cells produce ATP through aerobic glycolysis (the Warburg effect). Although this pattern exhibits low efficacy to produce ATP, the numerous mediates synthesized in this process can be used as building blocks that are more better for cell proliferation (Matthew G, et al., 2009, Science, 324:1029-1033).